[go: up one dir, main page]

EP1869088A2 - Compounds and methods for peptide synthesis - Google Patents

Compounds and methods for peptide synthesis

Info

Publication number
EP1869088A2
EP1869088A2 EP06748673A EP06748673A EP1869088A2 EP 1869088 A2 EP1869088 A2 EP 1869088A2 EP 06748673 A EP06748673 A EP 06748673A EP 06748673 A EP06748673 A EP 06748673A EP 1869088 A2 EP1869088 A2 EP 1869088A2
Authority
EP
European Patent Office
Prior art keywords
alkyl
cycloalkyl
aryl
heterocycloalkyl
independently
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06748673A
Other languages
German (de)
French (fr)
Inventor
Julio Herman Cuervo
Milka Yanachkova
Russell C. Petter
Thomas F. Durand-Reville
Jose Carlos Jimenez-Garcia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen Inc
Biogen MA Inc
Original Assignee
Biogen Idec Inc
Biogen Idec MA Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biogen Idec Inc, Biogen Idec MA Inc filed Critical Biogen Idec Inc
Publication of EP1869088A2 publication Critical patent/EP1869088A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers

Definitions

  • This invention relates to compounds and methods for peptide synthesis.
  • SPPS Solid phase peptide synthesis
  • the backbone nitrogen of a peptide can prevent aggregation during synthesis.
  • the backbone nitrogen can be modified by a group that interferes with the formation of hydrogen bonds involving the backbone nitrogen, which in turn can interfere with the formation of a ⁇ -sheet structure, ⁇ -sheet structures can sometimes lead to aggregation of peptides during synthesis.
  • including a backbone nitrogen modifying group can prevent or reduce the occurrence peptide aggregation during Fmoc- or Boc-based solid phase peptide synthesis.
  • the modifying group can disrupt formation of hydrogen bonds involving the backbone nitrogen.
  • the modifying group can enhance the aqueous solubility of a peptide, and facilitate purification and characterization of the peptide.
  • a method of making a peptide includes forming a peptide including a backbone nitrogen modifying group which includes a substituted aryl group.
  • the substituted aryl group includes a directing moiety and a hydrophilic moiety.
  • the peptide can be linked to a solid support.
  • the peptide can include at least one commonly occurring natural amino acid residue which optionally includes a protecting group.
  • the peptide can include at least one non-naturally occurring amino acid residue.
  • the method can include adding an amino acid residue to the peptide, thereby extending the peptide.
  • the method can include cleaving the peptide from the solid support without substantially removing the backbone nitrogen modifying group from the peptide.
  • the method can include removing the backbone nitrogen modifying group from the peptide.
  • the substituted aryl group can be a substituted phenyl group.
  • the substituted phenyl group can be ⁇ rt/z ⁇ -unsubstituted (i.e., unsubstituted in the 2- and 6-positions).
  • the hydrophilic moiety can include a tertiary amine.
  • the peptide can be substantially water-soluble.
  • the peptide can have the formula:
  • Each L is C 1 -C 10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, where L 1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NR d -, -NR d -C(0)-, -NR d C(O)NR d -, -OC(O)NR d -, -NR d -C(0)-0-, -S-, -S(O) 01 -, -NR d SO 2 -, -SO 2 NR d -, or -NR d -.
  • Each R c independently is -NR a R b , -OR a , -SR a , -S(O) m R a , -S(O) 2 NR 3 R 15 , -S(O) m OR a , -NR d C(0)R e , -O(CR d R e ) z NR a R b , -C(O)R a 5 -C(O)NR d R e , -NR a C(0)R b , -OC(O)NR a R b , -NR d C(0)0R a , -NR d C(O)NR a R b , heterocycloalkyl, or (heterocycloalkyl)alkyl.
  • R 2 can be optionally substituted with -I ⁇ R C .
  • Each R a is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fosed cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl.
  • Each R b is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fosed cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl.
  • Each R d is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl.
  • Each R e independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl.
  • Each A independently, is C 1 -C 10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene.
  • A optionally includes 1-3 heteroatoms selected from N, O and S.
  • Each R 3 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • Each R 3a is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • R 3 and R 3a together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, which optionally includes 1-6 heteroatoms selected from N, O, and S.
  • Each R 4 independently, is hydrogen or alkyl.
  • Each R 5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • Each R f and each R 8 independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group.
  • R 7 is hydrogen, alkyl, aryl, aralkyl, or a solid support.
  • Each i, and each j, independently, is zero or a positive integer, k is a positive integer, m is 1 or 2, n is 0, I 3 2, 3 or 4.
  • Each x, independently, is 1, 2, 3, 4, or 5, each y, independently, is 1, 2, 3, 4, or 5, and z is 1, 2, 3, 4, 5 or 6.
  • the peptide includes at least one -i ⁇ R 0 .
  • the peptide can have the formula:
  • X can be O.
  • L 1 can be C 1 -C 4 alkylene.
  • R c can be heterocycloalkyl.
  • -X-L'-R 0 can be 2-(morpholin-4-yl)ethoxy.
  • Each' R 5 independently, can be hydrogen or alkyl.
  • R 8 can be an amino protecting group.
  • Each A can be C 1 alkylene and each R 3a can be hydrogen.
  • the total of all i and all j can be less than 300.
  • the peptide can have a molecular weight of no greater than 40 kDa.
  • R 6 can be halo.
  • each j can be zero.
  • the method can include contacting the peptide with a compound having the formula:
  • R 4a can be hydrogen, alkyl, or an amino protecting group.
  • the compound can be 4-(2-(morpholin-4- yl)ethoxy)benzylamine.
  • the method can include adding an amino acid residue to the peptide, thereby forming a longer peptide.
  • the method can include contacting the peptide with a compound having the formula:
  • R 4a is hydrogen, alkyl, or an amino protecting group.
  • R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • R 5 is optionally substituted with -OR f , -SR f , -CO 2 R f , halo, haloalkyl, -CN, -NO 2 , -NR f R g ,
  • R 11 is hydroxy, alkoxy, aryloxy, aralkyloxy, or a leaving group, w is O 5 1 3 or 2.
  • a composition in another aspect, includes a peptide including a backbone nitrogen modifying group including a substituted aryl group, which includes a directing moiety and a hydrophilic moiety.
  • the peptide can include a plurality of backbone nitrogen modifying groups.
  • the peptide can have the formula:
  • R >4 4 a a can be hydrogen, alkyl, or an amino protecting group.
  • R 4a is hydrogen, alkyl, or an amino protecting group.
  • R 5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • R 11 is hydroxy, alkoxy, aryloxy, aralkyloxy, or a leaving group, w is 0, 1, or 2.
  • a method of making a peptide having a predetermined amino acid sequence includes determining a beta-sheet-forming propensity for at least a portion of the amino acid sequence, and selecting an amino acid residue of the sequence for modification with a backbone nitrogen modifying group based on the determined beta-sheet-forming propensity.
  • the backbone nitrogen modifying group can include a substituted aryl group which includes a directing moiety and a hydrophilic moiety.
  • ⁇ -sheet structure and concomitant peptide aggregation remains one of the most difficult challenges to overcome during solid phase peptide synthesis of peptides that contain regions capable of forming ⁇ -sheet structure or aggregating.
  • Most of the common available methods help to prevent aggregation in the early steps of SPPS, but these methods lack effectiveness in late steps in the synthesis of the peptide (e.g., during purification and characterization).
  • the peptide is removed from the solid support and deprotected, and instead of a pure, soluble product, an insoluble crude peptide which can be difficult to purify and characterize is obtained.
  • Current methods to prevent aggregation can also be incompatible with standard SPPS protocols.
  • a ⁇ sheet is made of several ⁇ -strands arranged side-by-side.
  • the peptide bonds in a ⁇ -strand adopt an almost fully extended conformation.
  • the side chains of adjacent amino acids in a ⁇ -strand point in opposite directions.
  • a ⁇ -sheet is formed by linking two or more ⁇ strands by hydrogen bonds. Adjacent chains in a ⁇ -sheet can run in opposite directions (an antiparallel ⁇ -sheet) or in the same direction (a parallel ⁇ -sheet).
  • the NH group and the CO group of each amino acid are respectively hydrogen bonded to the CO group and the NH group of a partner on the adjacent chain.
  • the hydrogen-bonding scheme is slightly more complicated.
  • the NH group is hydrogen bonded to the CO group of one amino acid on the adjacent strand, whereas the CO group is hydrogen bonded to the NH group on the amino acid two residues farther along the chain.
  • Many strands typically 4 or 5 but as many as 10 or more, can come together in ⁇ -sheets. Such ⁇ -sheets can be purely antiparallel, purely parallel, or mixed.
  • a region of a peptide is prone to forming ⁇ -sheet structure if it is known or believed to form a ⁇ -sheet structure.
  • hydrophobic residues are often found in beta-sheet structures.
  • the propensity of a particular region to form ⁇ -sheet structure can be predicted or estimated (see, for example, Smith, C.K., et at, Biochemistry 1994, 33(18):5510-7; and Creigton, T.E. Proteins, 2 nd ed., W.H. Freeman and Co., New York., 1993, each of which is incorporated by reference in its entirety).
  • a backbone nitrogen protecting group can block hydrogen bonding.
  • the protecting group is compatible with common SPPS conditions, contributes to aqueous solubility of the peptide, and is selectively removable when the synthesis is complete.
  • Selection of residues to be protected can be guided by a known secondary structure (e.g., as revealed by X-ray or NMR structural determination), an inferred secondary structure (e.g, based on sequence homology), or a predicted secondary structure.
  • a prediction or estimation of ⁇ -sheet forming propensity can guide the selection of residues to include a backbone nitrogen protecting group.
  • the backbone nitrogen of one or more residues in regions prone to forming ⁇ -sheet structures can be protected.
  • Solid supports used in solid phase peptide synthesis can include, for example, a Merrifield resin, a Wang resin, or a Rink resin.
  • a peptide is a compound including a peptide bond:
  • the peptide is formed by condensation of an amine with a carboxylic acid.
  • the peptide can be a polypeptide, in other words, including two or more peptide bonds.
  • the peptide can have a backbone (which includes the peptide bonds) and one or more side chains. In general the side chain is a substituent that branches from the backbone.
  • a peptide can be formed by the condensation of amino acids.
  • An amino acid is a compound including an amino group and a carboxylic acid group.
  • the amino acid can also include a side chain.
  • an amino acid can have the formula:
  • R represents the side chain.
  • the side chain of an amino acid is a substituent that branches from a backbone connecting the amino group to the carboxylic acid group.
  • the amino acid can be an alpha amino acid (as shown above, where the amino group is attached to the position alpha to the carboxylic acid group), a beta amino acid, a gamma amino acid, etc.
  • an amino acid ' residue refers to the portion of the peptide derived from a particular amino acid.
  • a polypeptide can be formed by sequential condensation of several amino acids.
  • a peptide can include any of the commonly naturally occurring amino acid residues (Ala, Cys, Asp, GIu, Phe, GIy, His, He, Lys, Leu, Met, Asn, Pro, GIn, Arg, Ser, Thr, VaI, Trp, and Tyr) in either stereochemical form (i.e., in D- or L- form).
  • a peptide can include other amino acid residues, such as a modified form of a common naturally occurring amino acid, or an unnatural amino acid.
  • non-naturally occurring amino acids are commercially available for use in SPPS, for example from Novabiochem, Sigma- Aldrich and other vendors.
  • protecting groups are linked to potentially reactive sites and prevent undesired reactions from occurring.
  • a protecting group can be removed selectively and completely.
  • protecting groups can be used for potentially reactive amino acid side chains, such as the side chains of Ser, Thr, Trp, Arg, Lys, Cys, His, Asp, GIu, Tyr, etc., and at the N- or C- terminus of the peptide.
  • the protecting groups can include, for example, tBoc, Fmoc, Cbz, Bz, etc. See, for example, Theodora W. Greene, Peter G. M. Wuts: Protective Groups in Organic Synthesis, 3 rd ed.
  • a hydroxyl group (as in the side chains of Ser and Thr) can be protected with, for example, a t-butyl group or a benzyl group.
  • Amino groups can be protected with, for example, Boc or Fmoc protecting groups.
  • Other protecting groups for reactive side chains are known.
  • the use of a backbone nitrogen protecting group can facilitate synthesis of difficult sequences. See, for example, Johnson, T., et al, J. Chem. Soc. Chem. Commun. 1993, 369-372; White, P., et al, J. Peptide Sci. 2004, 10, 18-26; and Johnson, T.
  • Backbone nitrogen protecting groups such as methyl, benzyl, and p-methylbenzyl can block ⁇ -sheet formation during routine solid phase peptide synthesis. This, in turn can block the aggregation induced by formation of hydrogen bonds, such as between ⁇ - strand structures.
  • these backbone nitrogen protecting groups can be difficult to remove by hydrogenation or HF, and do not promote solubility of the resulting peptides in aqueous buffers.
  • a 4-methoxy-substituted benzyl group When used as a backbone nitrogen protecting group, a 4-methoxy-substituted benzyl group can have higher acid lability can the corresponding 4-methyl-substituted benzyl group (see, for example, Johnson, T. and Quibell, M., Tetrahedron Lett. 1994, 35, 463-466).
  • a 4-methoxy-substituted benzyl group can be removed by HF or hydrogenation. It does not, however, effectively promote the solubility of the protected peptide fragments in aqueous buffers.
  • a backbone nitrogen modifying group can prevent formation of hydrogen bonds involving the backbone nitrogen of a peptide. This in turn prevents or reduces the extent of ⁇ -sheet formation during SPPS.
  • the modifying group preferably promotes the solubility of peptides in aqueous buffers and is compatible with standard SPPS protocols.
  • the peptide including the modifying group can be substantially water soluble, in other words, soluble in water, an aqueous buffer, or a water-solvent mixture.
  • the peptide can be soluble at concentrations of less than 10 mg/niL, less than 1 mg/niL, or less than 0.1 mg/mL.
  • a backbone modifying group can include a substituted aryl group, such as, for example, a substituted phenyl group.
  • the substituted aryl group can include a directing moiety and a hydrophilic moiety.
  • a directing moiety is a substituent on an aromatic ring that affects the reactivity of the ring, such as by increasing or decreasing reactivity at one or more positions on the ring.
  • a hydroxy (-OH) substituent can be a ⁇ r ⁇ -directing moiety (i.e., influencing reactivity at the position para to the hydroxy substituent), and a nitro (-NO 2 ) substituent can be a meto-directing moiety (i.e., influencing reactivity at the position meta to the hydroxy substituent).
  • the hydrophilic moiety can enhance the water solubility of a peptide including the modifying group.
  • the hydrophilic moiety can the water solubility of a peptide compared to a peptide lacking the hydrophilic moiety on a backbone nitrogen modifying group.
  • the directing moiety and the hydrophilic moiety can both belong to a single substituent on the aromatic ring.
  • the aryl group can optionally be ortho- unsubstituted.
  • the ort/zo-positions (i.e., the 2- and 6- positions) of the phenyl ring can be unsubstituted.
  • the aryl group can be r ⁇ et ⁇ -substituted,j? ⁇ r ⁇ -substituted, or both meta- and p ⁇ ra-substituted.
  • the modifying group can have the formula:
  • R 1 includes a directing moiety and R 2 includes a hydrophilic moiety; or R 1 includes a both directing moiety and a hydrophilic moiety.
  • the directing group can be, for example, an electron releasing group such as hydroxy, alkoxy, amino, alkylamino, or dialkylamino.
  • the directing group can be in apara position.
  • the hydrophilic moiety can include a heteroatom such as N, O, or S.
  • the hydrophilic moiety can include a hydrophilic group such as hydroxy or a tertiary amine.
  • the hydrophilic moiety can include a heterocyclic group, n can be 0, 1, 2, 3 or 4.
  • An aryl group is a cyclic aromatic group such as, for example, phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, or anthracenyl; or a heterocyclic aromatic group such as, for example, furyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, indolyl, benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzimidazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, or quinazolinyl.
  • a heterocyclyl group is a cyclic group including one or more heteroatoms in the ring, typically N, O 3 or S.
  • the heterocyclyl group can be monocyclic, bicyclic, tricyclic, or have four or more rings. When more than one ring is present, the rings can optionally be fused.
  • the heterocyclyl group can be an aryl group, an unsaturated group (i.e., including one or more double bonds), or a saturated group (i.e., including only single bonds).
  • a heterocycloalkyl group can be a saturated heterocylyl group.
  • heterocyclyl groups include tetrahydrofuryl, dihydrofuryl, furyl, oxazolyl, pyridyl, thioxazolyl, imidazolyl, benzimidazolyl, indolyl, pyrrolyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, and dioxanyl.
  • a backbone nitrogen modifying group can have the formula:
  • R 2 is optionally substituted with -L 1 -R c .
  • Each R a is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl.
  • Each R b is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl.
  • L 1 is C 1 -C 10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene. L 1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NR d -, -NR d -C(0)-, -NR d C(0)NR d -, -OC(O)NR d -, -NR d -C(O)-O-, -S-, -S(OV-, -NR d SO 2 -, -SO 2 NR d -, or -NR d -.
  • R c is -NR a R b , -OR a , -SR a , -S(O) m R a , -S(O) 2 NR a R b , -S(O) m OR a , -NR d C(0)R e , -0(CR d R e ) z NR a R b , -C(O)R a , -C(0)NR d R e , -NR a C(0)R b , -OC(O)NR a R b , -NR d C(0)0R a , -NR d C(O)NR a R b , heterocycloalkyl, or (heterocycloalkyl)alkyl.
  • Each R d independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloal
  • each R e independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl.
  • n is 0, 1, 2, 3 or 4
  • m is 1 or 2
  • z is 1, 2, 3, 4, 5 or 6.
  • the modifying group can have the formula:
  • R 2 , X, L 1 , R 0 and n are defined above.
  • the modifying group can be unsubstituted at the ⁇ rt/r ⁇ -positions.
  • the modifying group can have the formula:
  • the modifying group can be incorporated into a peptide.
  • the peptide can have the formula:
  • Each A 3 independently, is C 1 -C 10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene.
  • A optionally includes 1-3 heteroatoms selected from N, O and S.
  • Each R 3 independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • Each R 3a independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • R 3 and R 3a together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S;
  • Each R 4 independently, is hydrogen or alkyl.
  • R 3 and R 4 together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S.
  • Each R 5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • Each R independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group.
  • Each R g independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group.
  • R 8 is hydrogen, alkyl, an amino protecting group, or has the formula
  • Each i, independently, is zero or a positive integer.
  • Each j, independently, is zero or a positive integer. At least one j can be a positive integer, k is a positive integer; n is 0, 1, 2, 3 or 4; m is 1 or 2; each x, independently, is 1, 2, 3, 4, or 5; and each y, independently, is 1, 2, 3, 4, or 5.
  • X can be O, and L 1 can be C 1 -C 4 alkylene.
  • R 0 can be -NR a R b or heterocycloalkyl.
  • -X-L ⁇ R 0 can be 2-(morpholin-4-yl)ethoxy.
  • Each R 2 can be hydrogen.
  • A is C 1 alkylene
  • x and y can each be 1, and R a can be hydrogen.
  • each A is C 1 alkylene.
  • R is a solid support, R can be hydrogen or an amino protecting group.
  • each i can be, for example, less than 50, less than 30, less than 20, less than 10 or less than 5.
  • the sum of all i and j in the peptide can be, for example, less than 300, less than 200, less than 100, less than 75, less than 50, or less than 40.
  • the molecular weight of the peptide (excluding R 7 , if R 7 is a solid support) can be, for example, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 15 kDa, less than 10 kDa, or less than 5 kDa.
  • a method of making a peptide can include contacting a peptide having the formula:
  • R 2 , R 3 , R 3a , R 4 , R 5 , X, L 1 , R c , R 7 , i, j, k, and n are defined above, and R 6 is a leaving group; with a compound of formula:
  • R 1 , R 2 , R 4a and n are defined above.
  • the compound can have the formula:
  • the modifying group can be incorporated into an amino acid compound or an amino acid-derived compound.
  • the compound can have the formula:
  • Each R 3 and each R 3a independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
  • R 3 and R 4 together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S.
  • R 4a is hydrogen, alkyl, or an amino protecting group.
  • R 11 is hydroxy, alkoxy, aryloxy, aralkyloxy, a solid support, or a leaving group, w is 0, 1, or 2.
  • the amino acid or amino- acid derived compound can be used in solid phase peptide synthesis.
  • R 11 can form an activated ester with carbonyl group to which it is attached.
  • the activated ester can be, for example, a p-nitrophenyl ester, an N-hydroxysuccinimidyl ester, a pentafluorophenyl (OPfp) ester, a 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, a 1-hydroxybenzotriazole (HOBt) ester, or a l-hydroxy-7-azabenzotriazole (HOAt) ester.
  • a p-nitrophenyl ester an N-hydroxysuccinimidyl ester, a pentafluorophenyl (OPfp) ester, a 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, a 1-hydroxybenzotriazole (HOBt) ester, or a l-hydroxy-7-azabenzotriazole (HOAt) ester.
  • Ap ⁇ r ⁇ -methoxybenzyl group can be a backbone nitrogen modifying group.
  • the ⁇ r ⁇ -methoxy substituent can act as a directing group, increasing the reactivity of the modifying group compared to analogous benzyl or jr ⁇ r ⁇ -methylbenzyl modifying groups.
  • /? ⁇ r ⁇ -methoxybenzyl can be more acid labile as a backbone nitrogen modifying group than benzyl or/? ⁇ r ⁇ -methylbenzyl.
  • the par ⁇ -methoxybenzyl group is a poor water-solublizing moiety.
  • the methoxy group can be replaced with the more hydrophilic 2- (morpholin-4-yl)-ethoxy group.
  • the backbone nitrogen modifying group 4-(2- morpholin-4-yl-ethoxy) benzyl (MEB) has been synthesized and used in solid phase peptide synthesis.
  • the backbone nitrogen modifying group can be installed on a growing peptide chain during solid phase synthesis.
  • a protected amino acid can be added to a peptide chain by introducing an amino acid precursor to the growing chain.
  • the amino acid precursor can include a carbonyl group, and an alpha carbon.
  • the alpha carbon is linked to a side chain and to a leaving group.
  • the leaving group can be displaced by an amino group of a compound including the modifying group, for example, in a nucleophilic substitution. In this way, a backbone nitrogen-protected amino acid is added to the growing peptide chain.
  • the MEB modifying group can be installed on a peptide during solid phase synthesis.
  • the free amino terminus of a solid-support bound peptide is allowed to react with an amino acid precursor, such as, for example, chloroacetyl chloride.
  • the resulting chloroacetyl-substituted peptide can be reacted with MEBA, displacing the chloride leaving group.
  • the amino nitrogen of MEBA becomes a backbone nitrogen of the peptide.
  • Scheme 1 illustrates the addition of a MEB-protected amino acid to a growing peptide chain.
  • a polymer-bound peptide chain includes amino acid residues 1, 2, ..., n- ⁇ (with corresponding side chains indicated by R 1 , R 2 ,... , R n- i).
  • the subsequent amino acid residue to be introduced (residue ⁇ ), having a side chain indicated by R n is derived from a suitable precursor.
  • n can be less than 100, less than 75, less than 50, less than 40, less than 30, less than 20, or less than 10.
  • the precursor can include an activated carbonyl group, and an ⁇ -carbon.
  • the activated carbonyl group can be, for example, an acid halide such as an acid chloride or acid bromide, or an activated ester, such as, for example a succinimidyl ester, para-nitrophenyl ester, a pentafluorophenyl ester, or a 1-hydroxybenzotriazole ester.
  • an activated carbonyl group and other activated esters are known.
  • the activated carbonyl group includes a leaving group (shown as LG 1 ) linked to a carbonyl group.
  • R n and a leaving group (shown as LG 2 ) can be attached to the ⁇ -carbon.
  • the leaving group LG 2 can be, for example, a halo group.
  • the precursor can be chloroacetyl chloride or bromoacetic anhydride; if residue n is alanine (R n is methyl), the precursor can be 2-chloropropionyl chloride.
  • the precursor can be allowed to react with MEBA, thus adding the MEB-protected amino group of amino acid residue n.
  • the synthesis of the peptide chain can then continue using standard procedures. As shown in Scheme 1, amino acid residue n+ ⁇ is added by reaction with the MEB-protected amino group of amino acid residue n.
  • R n can be, for example, hydrogen or alkyl.
  • R n can be H, -CH 3 , -CH(CHs) 2 , -CH 2 CH(CH 3 ) 2 , or -CH(CH 3 )CH 2 CH 3 , such that the amino acid residue at position n can be GIy, Ala, VaI, Leu or He.
  • the MEB group can be first incorporated into an amino acid which is then added to a growing peptide chain during SPPS.
  • the carboxylic acid of a MEB-containing amino acid is coupled to the free amino group of a peptide linked to a solid support.
  • the MEB-containing amino acid can include an amino protecting group, such as Fmoc (as shown in Scheme 2) or Boc.
  • the MEB-containing amino acid can be deprotected to remove the amino protecting group, and a subsequent amino acid added to the peptide.
  • the MEB group can be first incorporated into a dipeptide which is then added to a growing peptide chain during SPPS.
  • the carboxylic acid of a MEB-containing dipeptide is coupled to the free amino group of a peptide linked to a solid support.
  • the MEB-containing dipeptide can include an amino protecting group, such as Fmoc (as shown in Scheme 3) or Boc.
  • the MEB-containing dipeptide can be deprotected to remove the amino protecting group, and a subsequent amino acid added to the peptide.
  • a long peptide e.g., a peptide of more than 40, more than 50, more than 75, more than 100, or 150 or more residues
  • the shorter peptides can be joined using native chemical ligation to afford the longer peptide (see, for example, Dawson, P.E. et ah, Science (1994) 266, 776, which is incorporated by reference in its entirety).
  • native chemical ligation allows the formation of a longer peptide from two shorter peptides, one having a C-terminal thioester (e.g., an aryl thioester), and the other peptide having an N-terminal cysteine residue.
  • both peptides should be water-soluble and highly pure. If one of the shorter peptides includes a difficult sequence, it can be prepared with a backbone nitrogen modifying group. The native chemical ligation can be performed prior to removal of the backbone nitrogen modifying group. The ligated peptide can assume its proper 3- dimensional fold after removal of the backbone nitrogen modifying group.
  • Method 1 The MEB group was added to a pre-selected sites (e.g., at a glycine residue) of a peptide sequence using alkylation with a resin-bound alpha-bromo carboxamide.
  • Bromoacetic anhydride was freshly prepared from bromoacetic acid (3.2 mmol, 444.64 mg).
  • the bromoacetic anhydride and diisopropylcarbodiimide (0.16 mmol, 0.025 mL) in chilled dichloromethane (12 mL) was added to the N-terminus of a resin- supported growing peptide chain (0.16 mmol, 400 mg).
  • the peptide was synthesized using standard Fmoc protocols.
  • Method 2 In this method the pre-selected sites of the peptide sequence were modified with a dipeptide unit (see below) in which the amide bond has been protected with a MEB group. The protected dipeptide unit was then coupled to the N-terminus of a growing peptide and the synthesis was continued using standard Fmoc protocols. Preparation of a MEB-protected dipeptide. To a bromoacetic acid pre-loaded HMP-resin (1 mmol, 2.5 g), MEBA (5.2 mmol, 1.23 g) in dichloromethane (10 niL) was added and the mixture was shaken at 20 ° C for 20 hours. The mixture was filtered using a 50 niL polypropylene filtration tube.
  • the resin was washed with dichloromethane (4 x 10 mL).
  • a mixture of N-alpha-Fmoc-protected amino acid (5 mmol) in N,N- dimethylformamide (20 mL), bromotripyrrolidinophosphonium hexafluorophosphate (4.8 mmol, 2.24 g) and N,N-diisopropylethylarnine (10 mmol, 1.74 mL) were mixed for 5 minutes at 20 °C before adding to the resin.
  • the resin was shaken at 20 0 C for 18 hours.
  • the resin was then washed with N,N-dimethylformamide (4 x 10 mL) and dichloromethane (2 x 10 mL).
  • the resin was dried in vacuo.
  • the dipeptide was cleaved from the resin with trifluoroacetic acid/water, 9/1 (10.0 mL) at 20 0 C for 2 hours.
  • the trifluoroacetic acid-resin mixture was filtered to remove the resin.
  • Trifluoroacetic acid was removed under reduced pressure to give the MEB-protected dipeptide unit.
  • the dipeptide can then be used in SPPS of a longer peptide.
  • a 50-mL Teflon tube containing a mixture of MEBA-modified peptide (0.018 mmol, 25 mg) and p-cresol (400 mg) was mounted onto an HF apparatus.
  • the tube was immersed into a dry ice acetone bath and anhydrous hydrogen fluoride (10 mL) was condensed.
  • the dry ice acetone bath was replaced by a water bath containing crushed ice, and the reaction was magnetically stirred for 1 hour.
  • the hydrogen fluoride was evaporated from the Teflon tube and trapped into a 15% solution of potassium hydroxide using nitrogen gas at 20 psi for 30 minutes.
  • the Teflon tube was removed from the HF apparatus and the peptide was precipitated with chilled ethyl ether.
  • the solid peptide was then taken up with a 50% solution of acetonitrile-water.
  • the solution was frozen and lyophilized to give the MEB-deprotected peptide.
  • Table 1 summarizes the results of experiments testing the removal of backbone nitrogen modifying groups under various conditions.
  • CEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLLF AESGQ VYFGIIAL corresponds to the C-terminus of human TNF-alpha.
  • the 48-amino acid peptide was prepared by solid phase peptide synthesis using Fmoc-protected amino acids on an Applied Biosystems 433 A peptide synthesizer according to manufacture specific protocols.
  • a commercial available Fmoc-Leu-Wang Resin (0.47 g.
  • Fmoc-amino acids were activated by the addition of equimolar amounts of HBTU and HOBt and 2 equivalents of DIEA in DMF.
  • Three MEB groups were introduced at Gl 3, G40 and G45 using method 1.
  • the peptide was cleaved from the resin and deprotected with TFA/EDT/TA/phenol/water/TIPS (68.5:10:10:5:3.5:1 V: V). The TFA resin mixture was filtered.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

A backbone nitrogen modifying group can prevent aggregation of peptides during peptide synthesis. The modifying group can promote aqueous solubility of the peptides, and be compatible with solid phase peptide synthesis. Methods for making peptides are also described.

Description

COMPOUNDS AND METHODS FOR PEPTIDE
SYNTHESIS
CLAIM OF PRIORITY
This application claims priority under 35 USC §119(e) to U.S. Patent Application Serial No. 60/664,945 filed on March 28, 2005, which is incorporated by reference in its entirety.
TECHNICAL FIELD
This invention relates to compounds and methods for peptide synthesis.
BACKGROUND
Solid phase peptide synthesis (SPPS) can be used to prepare a wide variety of peptide sequences. Some sequences, however, can be difficult to prepare by standard methods of SPPS. These difficult sequences are often characterized by a high content of hydrophobic amino acids and a tendency to adopt a β-sheet secondary structure. Peptides that have a difficult sequence can aggregate during synthesis. Aggregation can have a negative impact on the yield, purity, and water solubility of the final peptide product.
SUMMARY
Protecting the backbone nitrogen of a peptide can prevent aggregation during synthesis. The backbone nitrogen can be modified by a group that interferes with the formation of hydrogen bonds involving the backbone nitrogen, which in turn can interfere with the formation of a β-sheet structure, β-sheet structures can sometimes lead to aggregation of peptides during synthesis. In particular, including a backbone nitrogen modifying group can prevent or reduce the occurrence peptide aggregation during Fmoc- or Boc-based solid phase peptide synthesis. The modifying group can disrupt formation of hydrogen bonds involving the backbone nitrogen. The modifying group can enhance the aqueous solubility of a peptide, and facilitate purification and characterization of the peptide. The modifying group can be compatible with reactions conditions used in Fmoc and Boc synthesis, and does not interfere with chemical peptide ligation. Desirably, the modifying group can be easily removed from the final peptide product. In one aspect, a method of making a peptide includes forming a peptide including a backbone nitrogen modifying group which includes a substituted aryl group. The substituted aryl group includes a directing moiety and a hydrophilic moiety.
The peptide can be linked to a solid support. The peptide can include at least one commonly occurring natural amino acid residue which optionally includes a protecting group. The peptide can include at least one non-naturally occurring amino acid residue.
The method can include adding an amino acid residue to the peptide, thereby extending the peptide. The method can include cleaving the peptide from the solid support without substantially removing the backbone nitrogen modifying group from the peptide. The method can include removing the backbone nitrogen modifying group from the peptide.
The substituted aryl group can be a substituted phenyl group. The substituted phenyl group can be ørt/zø-unsubstituted (i.e., unsubstituted in the 2- and 6-positions). The hydrophilic moiety can include a tertiary amine. The peptide can be substantially water-soluble.
The peptide can have the formula:
X is O, S, NH, or a bond. Each L , independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, where L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(0)-, -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(0)-0-, -S-, -S(O)01-, -NRdSO2-, -SO2NRd-, or -NRd-. Each Rc, independently is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NR3R15, -S(O)mORa, -NRdC(0)Re, -O(CRdRe)zNRaRb, -C(O)Ra 5 -C(O)NRdRe, -NRaC(0)Rb, -OC(O)NRaRb, -NRdC(0)0Ra, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl.
Each R2, independently, is hydrogen, -Ra, -0Ra, -SRa, -NRaRb, -NRaC(=O)Rb, or halo. R2 can be optionally substituted with -IΛRC. Each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fosed cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl. Each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fosed cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl.
Each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl. Each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl.
Each R9, independently, is hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, or halo; and each R10, independently, is hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, or halo.
Each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene. A optionally includes 1-3 heteroatoms selected from N, O and S.
Each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R3 is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfR8, or -NHC(=NH)NRfRg. Each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R3a is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg. R3 and R3a together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, which optionally includes 1-6 heteroatoms selected from N, O, and S.
Each R4, independently, is hydrogen or alkyl. R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, which optionally includes 1-6 heteroatoms selected from N, O, and S.
Each R5, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R5 is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg. Each Rf and each R8, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group.
R7 is hydrogen, alkyl, aryl, aralkyl, or a solid support. R8 is hydrogen, alkyl, an amino protecting group, or has the formula -C(=O)CH(R5)R6, where R6 is a leaving group.
Each i, and each j, independently, is zero or a positive integer, k is a positive integer, m is 1 or 2, n is 0, I3 2, 3 or 4. Each x, independently, is 1, 2, 3, 4, or 5, each y, independently, is 1, 2, 3, 4, or 5, and z is 1, 2, 3, 4, 5 or 6.
In certain circumstances, the peptide includes at least one -iΛR0. The peptide can have the formula:
X can be O. L1 can be C1-C4 alkylene. Rc can be heterocycloalkyl. In particular, -X-L'-R0 can be 2-(morpholin-4-yl)ethoxy. Each' R5, independently, can be hydrogen or alkyl. When R7 is a solid support, R8 can be an amino protecting group. Each A can be C1 alkylene and each R3a can be hydrogen. The total of all i and all j can be less than 300. The peptide can have a molecular weight of no greater than 40 kDa. R8 can have the formula -C(=O)CH(RS)R6, where R6 is a leaving group. R6 can be halo. When used in the method, each j can be zero.
The method can include contacting the peptide with a compound having the formula:
where X, L1, Rc, R2 and n are defined above. In the compound, R4a can be hydrogen, alkyl, or an amino protecting group. The compound can be 4-(2-(morpholin-4- yl)ethoxy)benzylamine. The method can include adding an amino acid residue to the peptide, thereby forming a longer peptide.
The method can include contacting the peptide with a compound having the formula:
or
where X, L1, Rc, R2, R3, R3a, R4, R5, x, y, and n are defined above. In the compound, R4a is hydrogen, alkyl, or an amino protecting group. R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl.
R5 is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg,
-C(=O)NRfRg, or -NHC(=NH)NRfRs. R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, or a leaving group, w is O5 13 or 2.
In another aspect, a composition includes a peptide including a backbone nitrogen modifying group including a substituted aryl group, which includes a directing moiety and a hydrophilic moiety. The peptide can include a plurality of backbone nitrogen modifying groups.
The peptide can have the formula:
as described above.
In another aspect, a compound having the formula:
where X, L1, Rc, R2 and n are defined above. In the compound, R >44aa can be hydrogen, alkyl, or an amino protecting group.
In another aspect, a compound having the formula:
or
where X, L1, Rc, R2, R4, and n are defined above. In the compound, R4a is hydrogen, alkyl, or an amino protecting group. R5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R5 is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRε, -C(=O)NRfRg, or -NHC(=NH)NRfRg. R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, or a leaving group, w is 0, 1, or 2.
In yet another aspect, a method of making a peptide having a predetermined amino acid sequence includes determining a beta-sheet-forming propensity for at least a portion of the amino acid sequence, and selecting an amino acid residue of the sequence for modification with a backbone nitrogen modifying group based on the determined beta-sheet-forming propensity. The backbone nitrogen modifying group can include a substituted aryl group which includes a directing moiety and a hydrophilic moiety.
The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description, and from the claims.
DETAILED DESCRIPTION
The formation of β-sheet structure and concomitant peptide aggregation remains one of the most difficult challenges to overcome during solid phase peptide synthesis of peptides that contain regions capable of forming β-sheet structure or aggregating. Most of the common available methods help to prevent aggregation in the early steps of SPPS, but these methods lack effectiveness in late steps in the synthesis of the peptide (e.g., during purification and characterization). In the late steps, the peptide is removed from the solid support and deprotected, and instead of a pure, soluble product, an insoluble crude peptide which can be difficult to purify and characterize is obtained. Current methods to prevent aggregation can also be incompatible with standard SPPS protocols. A β sheet is made of several β-strands arranged side-by-side. The peptide bonds in a β-strand adopt an almost fully extended conformation. The side chains of adjacent amino acids in a β-strand point in opposite directions. A β-sheet is formed by linking two or more β strands by hydrogen bonds. Adjacent chains in a β-sheet can run in opposite directions (an antiparallel β-sheet) or in the same direction (a parallel β-sheet). In the antiparallel arrangement, the NH group and the CO group of each amino acid are respectively hydrogen bonded to the CO group and the NH group of a partner on the adjacent chain. In the parallel arrangement, the hydrogen-bonding scheme is slightly more complicated. For each amino acid, the NH group is hydrogen bonded to the CO group of one amino acid on the adjacent strand, whereas the CO group is hydrogen bonded to the NH group on the amino acid two residues farther along the chain. Many strands, typically 4 or 5 but as many as 10 or more, can come together in β-sheets. Such β-sheets can be purely antiparallel, purely parallel, or mixed.
A region of a peptide is prone to forming β-sheet structure if it is known or believed to form a β-sheet structure. For example, hydrophobic residues are often found in beta-sheet structures. The propensity of a particular region to form β-sheet structure can be predicted or estimated (see, for example, Smith, C.K., et at, Biochemistry 1994, 33(18):5510-7; and Creigton, T.E. Proteins, 2nd ed., W.H. Freeman and Co., New York., 1993, each of which is incorporated by reference in its entirety).
When a synthetic peptide forms a β-sheet structure during synthesis, it can cause aggregation of the peptides, leading to undesirable results such as poor yield or insoluble products. Because β-sheets rely on hydrogen bonding between backbone nitrogen and carbonyl oxygen atoms, the formation of β-sheets can be prevented by blocking hydrogen bonding at these positions. A backbone nitrogen protecting group can block hydrogen bonding. Preferably, the protecting group is compatible with common SPPS conditions, contributes to aqueous solubility of the peptide, and is selectively removable when the synthesis is complete. Selection of residues to be protected can be guided by a known secondary structure (e.g., as revealed by X-ray or NMR structural determination), an inferred secondary structure (e.g, based on sequence homology), or a predicted secondary structure. A prediction or estimation of β-sheet forming propensity can guide the selection of residues to include a backbone nitrogen protecting group. In particular, the backbone nitrogen of one or more residues in regions prone to forming β-sheet structures can be protected.
Solid phase peptide synthesis is described in, for example, Weng C. Chan, and Peter D. White: Fmoc Solid Phase Peptide Synthesis: A Practical Approach, 2000, Oxford University Press; and John Jones, Amino Acid and Peptide Synthesis, 2002,
Oxford University Press, each of which is incorporated by reference in its entirety. Solid supports used in solid phase peptide synthesis can include, for example, a Merrifield resin, a Wang resin, or a Rink resin.
In general, a peptide is a compound including a peptide bond:
O Typically, the peptide is formed by condensation of an amine with a carboxylic acid. The peptide can be a polypeptide, in other words, including two or more peptide bonds. The peptide can have a backbone (which includes the peptide bonds) and one or more side chains. In general the side chain is a substituent that branches from the backbone. A peptide can be formed by the condensation of amino acids. An amino acid is a compound including an amino group and a carboxylic acid group. The amino acid can also include a side chain. For example, an amino acid can have the formula:
R
H2N^CO2H where R represents the side chain. In general the side chain of an amino acid is a substituent that branches from a backbone connecting the amino group to the carboxylic acid group. The amino acid can be an alpha amino acid (as shown above, where the amino group is attached to the position alpha to the carboxylic acid group), a beta amino acid, a gamma amino acid, etc. When a peptide is formed by the condensation of amino acids, the peptide can be described as including an amino acid residue. An amino acid ' residue refers to the portion of the peptide derived from a particular amino acid. For
example, the residue that results when the amino acid alanine, , is
incorporated into a peptide is: . A polypeptide can be formed by sequential condensation of several amino acids.
A peptide can include any of the commonly naturally occurring amino acid residues (Ala, Cys, Asp, GIu, Phe, GIy, His, He, Lys, Leu, Met, Asn, Pro, GIn, Arg, Ser, Thr, VaI, Trp, and Tyr) in either stereochemical form (i.e., in D- or L- form). A peptide can include other amino acid residues, such as a modified form of a common naturally occurring amino acid, or an unnatural amino acid. A wide variety of non-naturally occurring amino acids are commercially available for use in SPPS, for example from Novabiochem, Sigma- Aldrich and other vendors.
It can be desirable to use protecting groups during SPPS. The protecting groups are linked to potentially reactive sites and prevent undesired reactions from occurring. Preferably, a protecting group can be removed selectively and completely. In SPPS, protecting groups can be used for potentially reactive amino acid side chains, such as the side chains of Ser, Thr, Trp, Arg, Lys, Cys, His, Asp, GIu, Tyr, etc., and at the N- or C- terminus of the peptide. The protecting groups can include, for example, tBoc, Fmoc, Cbz, Bz, etc. See, for example, Theodora W. Greene, Peter G. M. Wuts: Protective Groups in Organic Synthesis, 3 rd ed. Wiley Interscience, 1999, which is incorporated by reference in its entirety. For example, a hydroxyl group (as in the side chains of Ser and Thr) can be protected with, for example, a t-butyl group or a benzyl group. Amino groups can be protected with, for example, Boc or Fmoc protecting groups. Other protecting groups for reactive side chains are known. The use of a backbone nitrogen protecting group can facilitate synthesis of difficult sequences. See, for example, Johnson, T., et al, J. Chem. Soc. Chem. Commun. 1993, 369-372; White, P., et al, J. Peptide Sci. 2004, 10, 18-26; and Johnson, T. and Quibell, M., Tetrahedron Lett. 1994, 35, 463-466, each which is incorporated by reference in its entirety. Backbone nitrogen protecting groups such as methyl, benzyl, and p-methylbenzyl can block β-sheet formation during routine solid phase peptide synthesis. This, in turn can block the aggregation induced by formation of hydrogen bonds, such as between β- strand structures. However, these backbone nitrogen protecting groups can be difficult to remove by hydrogenation or HF, and do not promote solubility of the resulting peptides in aqueous buffers.
When used as a backbone nitrogen protecting group, a 4-methoxy-substituted benzyl group can have higher acid lability can the corresponding 4-methyl-substituted benzyl group (see, for example, Johnson, T. and Quibell, M., Tetrahedron Lett. 1994, 35, 463-466). A 4-methoxy-substituted benzyl group can be removed by HF or hydrogenation. It does not, however, effectively promote the solubility of the protected peptide fragments in aqueous buffers.
A backbone nitrogen modifying group can prevent formation of hydrogen bonds involving the backbone nitrogen of a peptide. This in turn prevents or reduces the extent of β-sheet formation during SPPS. The modifying group preferably promotes the solubility of peptides in aqueous buffers and is compatible with standard SPPS protocols. The peptide including the modifying group can be substantially water soluble, in other words, soluble in water, an aqueous buffer, or a water-solvent mixture. The peptide can be soluble at concentrations of less than 10 mg/niL, less than 1 mg/niL, or less than 0.1 mg/mL.
In general, a backbone modifying group can include a substituted aryl group, such as, for example, a substituted phenyl group. The substituted aryl group can include a directing moiety and a hydrophilic moiety. A directing moiety is a substituent on an aromatic ring that affects the reactivity of the ring, such as by increasing or decreasing reactivity at one or more positions on the ring. For example, a hydroxy (-OH) substituent can be a^αrα-directing moiety (i.e., influencing reactivity at the position para to the hydroxy substituent), and a nitro (-NO2) substituent can be a meto-directing moiety (i.e., influencing reactivity at the position meta to the hydroxy substituent). The hydrophilic moiety can enhance the water solubility of a peptide including the modifying group. In particular, the hydrophilic moiety can the water solubility of a peptide compared to a peptide lacking the hydrophilic moiety on a backbone nitrogen modifying group. In certain embodiments, the directing moiety and the hydrophilic moiety can both belong to a single substituent on the aromatic ring. The aryl group can optionally be ortho- unsubstituted. For example, if the modifying group is a substituted benzyl group, the ort/zo-positions (i.e., the 2- and 6- positions) of the phenyl ring can be unsubstituted. The aryl group can be røetα-substituted,j?αrø-substituted, or both meta- and pαra-substituted. The modifying group can have the formula:
where Ar is an aryl group, R1 includes a directing moiety and R2 includes a hydrophilic moiety; or R1 includes a both directing moiety and a hydrophilic moiety. The directing group can be, for example, an electron releasing group such as hydroxy, alkoxy, amino, alkylamino, or dialkylamino. The directing group can be in apara position. The hydrophilic moiety can include a heteroatom such as N, O, or S. The hydrophilic moiety can include a hydrophilic group such as hydroxy or a tertiary amine. The hydrophilic moiety can include a heterocyclic group, n can be 0, 1, 2, 3 or 4.
An aryl group is a cyclic aromatic group such as, for example, phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, or anthracenyl; or a heterocyclic aromatic group such as, for example, furyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, indolyl, benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzimidazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, or quinazolinyl. A heterocyclyl group is a cyclic group including one or more heteroatoms in the ring, typically N, O3 or S. The heterocyclyl group can be monocyclic, bicyclic, tricyclic, or have four or more rings. When more than one ring is present, the rings can optionally be fused. The heterocyclyl group can be an aryl group, an unsaturated group (i.e., including one or more double bonds), or a saturated group (i.e., including only single bonds). A heterocycloalkyl group can be a saturated heterocylyl group. Some examples of heterocyclyl groups include tetrahydrofuryl, dihydrofuryl, furyl, oxazolyl, pyridyl, thioxazolyl, imidazolyl, benzimidazolyl, indolyl, pyrrolyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, and dioxanyl. A backbone nitrogen modifying group can have the formula:
where:
R1 is -Ra, -ORa, -SR\ -NRaRb, -NRaC(=O)Rb, halo, or -X-lΛRc. Each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo. R2 is optionally substituted with -L1 -Rc.
Each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl. Each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl.
X is O, S, NH, or a bond. L1 is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene. L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(0)-, -NRdC(0)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(OV-, -NRdSO2-, -SO2NRd-, or -NRd-. Rc is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(0)Re, -0(CRdRe)zNRaRb, -C(O)Ra, -C(0)NRdRe, -NRaC(0)Rb, -OC(O)NRaRb, -NRdC(0)0Ra, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl. Each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl,
(heterocycloalkyl)alkyl, or aryl. Each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl. n is 0, 1, 2, 3 or 4, m is 1 or 2, and z is 1, 2, 3, 4, 5 or 6. In some circumstances, the modifying group can have the formula:
where R2, X, L1, R0 and n are defined above. The modifying group can be unsubstituted at the ørt/rø-positions.
The modifying group can have the formula:
The modifying group can be incorporated into a peptide. The peptide can have the formula:
or
where R2, X, L1, Rc, R7, R9, and R10,are defined above. Each A3 independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene. A optionally includes 1-3 heteroatoms selected from N, O and S. Each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R3 is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg. Each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R3a is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg. R3 and R3a, together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S;
Each R4, independently, is hydrogen or alkyl. R3 and R4 together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S.
Each R5, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R5 is optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg.
Each R , independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group. Each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group. R8 is hydrogen, alkyl, an amino protecting group, or has the formula
-C(=O)CH(R5)R6, where R6 is a leaving group.
Each i, independently, is zero or a positive integer. Each j, independently, is zero or a positive integer. At least one j can be a positive integer, k is a positive integer; n is 0, 1, 2, 3 or 4; m is 1 or 2; each x, independently, is 1, 2, 3, 4, or 5; and each y, independently, is 1, 2, 3, 4, or 5.
In the peptide, X can be O, and L1 can be C1-C4 alkylene. R0 can be -NRaRb or heterocycloalkyl. In particular, -X-L^R0 can be 2-(morpholin-4-yl)ethoxy. Each R2 can be hydrogen. When A is C1 alkylene, x and y can each be 1, and R a can be hydrogen. In some circumstances, each A is C1 alkylene. When R is a solid support, R can be hydrogen or an amino protecting group. In the peptide, each i can be, for example, less than 50, less than 30, less than 20, less than 10 or less than 5. The sum of all i and j in the peptide can be, for example, less than 300, less than 200, less than 100, less than 75, less than 50, or less than 40. The molecular weight of the peptide (excluding R7, if R7 is a solid support) can be, for example, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 15 kDa, less than 10 kDa, or less than 5 kDa.
A method of making a peptide can include contacting a peptide having the formula:
where R2, R3, R3a, R4, R5, X, L1, Rc, R7, i, j, k, and n are defined above, and R6 is a leaving group; with a compound of formula:
where R1, R2, R4a and n are defined above.
In some circumstances the compound can have the formula:
where X5 L1, Rc, R2, R4a and n are defined above.
The modifying group can be incorporated into an amino acid compound or an amino acid-derived compound. The compound can have the formula:
or
where X5 L1, Rc, R4, R5 and R2 are defined above. Each R3 and each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl. R3 and R3a are each independently optionally substituted with -ORf, -SRf 3 -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfR8, -C(=O)NRf, -NHC(=NH)NRfRs;R4 is hydrogen, alkyl, or an amino protecting group. R3 and R4 together with the atoms to which they are attached can form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S. R4a is hydrogen, alkyl, or an amino protecting group. R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, a solid support, or a leaving group, w is 0, 1, or 2. The amino acid or amino- acid derived compound can be used in solid phase peptide synthesis. When R11 is a leaving group, R11 can form an activated ester with carbonyl group to which it is attached. The activated ester can be, for example, a p-nitrophenyl ester, an N-hydroxysuccinimidyl ester, a pentafluorophenyl (OPfp) ester, a 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, a 1-hydroxybenzotriazole (HOBt) ester, or a l-hydroxy-7-azabenzotriazole (HOAt) ester.
Apαrα-methoxybenzyl group can be a backbone nitrogen modifying group. The ^αrα-methoxy substituent can act as a directing group, increasing the reactivity of the modifying group compared to analogous benzyl or jrørø-methylbenzyl modifying groups. For example, /?αrø-methoxybenzyl can be more acid labile as a backbone nitrogen modifying group than benzyl or/?αrα-methylbenzyl. However, the par α-methoxybenzyl group is a poor water-solublizing moiety. To increase water solubility without compromising acid lability of a 4- methoxybenzyl group, the methoxy group can be replaced with the more hydrophilic 2- (morpholin-4-yl)-ethoxy group. The backbone nitrogen modifying group 4-(2- morpholin-4-yl-ethoxy) benzyl (MEB) has been synthesized and used in solid phase peptide synthesis.
The backbone nitrogen modifying group can be installed on a growing peptide chain during solid phase synthesis. A protected amino acid can be added to a peptide chain by introducing an amino acid precursor to the growing chain. The amino acid precursor can include a carbonyl group, and an alpha carbon. The alpha carbon is linked to a side chain and to a leaving group. The leaving group can be displaced by an amino group of a compound including the modifying group, for example, in a nucleophilic substitution. In this way, a backbone nitrogen-protected amino acid is added to the growing peptide chain.
For example, the MEB modifying group can be installed on a peptide during solid phase synthesis. The free amino terminus of a solid-support bound peptide is allowed to react with an amino acid precursor, such as, for example, chloroacetyl chloride. The resulting chloroacetyl-substituted peptide can be reacted with MEBA, displacing the chloride leaving group. The amino nitrogen of MEBA becomes a backbone nitrogen of the peptide.
Scheme 1 illustrates the addition of a MEB-protected amino acid to a growing peptide chain. In Scheme 1, a polymer-bound peptide chain includes amino acid residues 1, 2, ..., n-\ (with corresponding side chains indicated by R1, R2,... , Rn-i). The subsequent amino acid residue to be introduced (residue ή), having a side chain indicated by Rn, is derived from a suitable precursor. For example, n can be less than 100, less than 75, less than 50, less than 40, less than 30, less than 20, or less than 10. The precursor can include an activated carbonyl group, and an α-carbon. The activated carbonyl group can be, for example, an acid halide such as an acid chloride or acid bromide, or an activated ester, such as, for example a succinimidyl ester, para-nitrophenyl ester, a pentafluorophenyl ester, or a 1-hydroxybenzotriazole ester. Other activated carbonyl group and other activated esters are known. In general, the activated carbonyl group includes a leaving group (shown as LG1) linked to a carbonyl group. Rn and a leaving group (shown as LG2) can be attached to the α-carbon. The leaving group LG2 can be, for example, a halo group. For example, if residue n is glycine (Rn is hydrogen), the precursor can be chloroacetyl chloride or bromoacetic anhydride; if residue n is alanine (Rn is methyl), the precursor can be 2-chloropropionyl chloride. Once the precursor has been added to the growing peptide chain, the resulting peptide can be allowed to react with MEBA, thus adding the MEB-protected amino group of amino acid residue n. The synthesis of the peptide chain can then continue using standard procedures. As shown in Scheme 1, amino acid residue n+\ is added by reaction with the MEB-protected amino group of amino acid residue n.
Scheme 1
Rn can be, for example, hydrogen or alkyl. Rn can be H, -CH3, -CH(CHs)2, -CH2CH(CH3)2, or -CH(CH3)CH2CH3, such that the amino acid residue at position n can be GIy, Ala, VaI, Leu or He.
Alternatively, the MEB group can be first incorporated into an amino acid which is then added to a growing peptide chain during SPPS. As shown in Scheme 2, the carboxylic acid of a MEB-containing amino acid is coupled to the free amino group of a peptide linked to a solid support. The MEB-containing amino acid can include an amino protecting group, such as Fmoc (as shown in Scheme 2) or Boc. The MEB-containing amino acid can be deprotected to remove the amino protecting group, and a subsequent amino acid added to the peptide. Scheme 2
deprotection
In another method, the MEB group can be first incorporated into a dipeptide which is then added to a growing peptide chain during SPPS. As shown in Scheme 3, the carboxylic acid of a MEB-containing dipeptide is coupled to the free amino group of a peptide linked to a solid support. The MEB-containing dipeptide can include an amino protecting group, such as Fmoc (as shown in Scheme 3) or Boc. The MEB-containing dipeptide can be deprotected to remove the amino protecting group, and a subsequent amino acid added to the peptide. Scheme 3
In some circumstances, it can be desirable to prepare a long peptide (e.g., a peptide of more than 40, more than 50, more than 75, more than 100, or 150 or more residues) by first synthesizing two (or more) shorter peptides. The shorter peptides can be joined using native chemical ligation to afford the longer peptide (see, for example, Dawson, P.E. et ah, Science (1994) 266, 776, which is incorporated by reference in its entirety). In general, native chemical ligation allows the formation of a longer peptide from two shorter peptides, one having a C-terminal thioester (e.g., an aryl thioester), and the other peptide having an N-terminal cysteine residue. In order to perform native chemical ligation, both peptides should be water-soluble and highly pure. If one of the shorter peptides includes a difficult sequence, it can be prepared with a backbone nitrogen modifying group. The native chemical ligation can be performed prior to removal of the backbone nitrogen modifying group. The ligated peptide can assume its proper 3- dimensional fold after removal of the backbone nitrogen modifying group.
Examples
Synthesis of MEB A
4-(2~morpholinoethoxy)benzonitrile: 6.1 g (0.051 mol) of 4-cyanophenol (Aldrich) was combined with 14.3 g (0.0768 mol) of 4-(2-chloroethyl)morpholine hydrochloride (Aldrich), 21.1 g (0.153 mol) of potassium carbonate and 2 g of potassium iodide in 250 mL of anhydrous dimethylformamide (DMF) and heated at 60 °C for 4 hours. The reaction was monitored by LC/MS and TLC (EtOAc/hexane 1 : 1 or 100% EtOAc). The TLC plate was deactivated with 1% triethylarriine (TEA) in hexane. After the completion of the reaction the reaction mixture was filtered and the precipitate washed three times with 10 mL of DMF. The filtrate was concentrated by rotary evaporator. The residue was dissolved in dichloromethane and filtered through silica pretreated with 1% TEA in 10% EtOAc/hexane, and rinsed with ethyl acetate. Yield: 9.6 g (81%) of 4-(2-morpholinoethoxy)benzonitrile compound as white to slightly pink needles.
4-(2-morpholinoethoxy)benzylamine (MEBA): 10 mL of a 1.0 M solution of LiAlH4 in tetrahydrofuran (THF) was added dropwise to a stirred, cooled (0-4 °C) solution of 1.15 g of 4-(2-morpholinoethoxy)benzonitrile in anhydrous THF. After the addition was completed, the reaction was allowed to warm to room temperature. The reaction was monitored by LC/MS for disappearance of the starting material and also for the aldehyde side product (M+H = 236). The presence of the aldehyde side product can indicate an incomplete reduction, as the aldehyde forms from unreacted imine during analysis. After 6-8 hours, the reaction mixture was added slowly to a stirred, cooled solution of saturated NH4Cl (50 rnL), stirred for 30 minutes, and filtered. The filtrate was extracted with a 25 mL portion of ethyl acetate. The aqueous phase was separated and a portion of 50% NaOH half the volume of the aqueous phase was added. This mixture was extracted twice with 50 mL of dioxane. Combined dioxane extracts were stirred for 30 minutes with solid potassium hydroxide (~1 g) to remove water. The potassium hydroxide slurry was removed and the solvent removed by rotary evaporator. The resulting colorless oil crystallized when allowed to stand at 4 0C. Yield: 980 mg, 84%.
Introduction of a MEB group into a peptide
Method 1. The MEB group was added to a pre-selected sites (e.g., at a glycine residue) of a peptide sequence using alkylation with a resin-bound alpha-bromo carboxamide. Bromoacetic anhydride was freshly prepared from bromoacetic acid (3.2 mmol, 444.64 mg). The bromoacetic anhydride and diisopropylcarbodiimide (0.16 mmol, 0.025 mL) in chilled dichloromethane (12 mL) was added to the N-terminus of a resin- supported growing peptide chain (0.16 mmol, 400 mg). The peptide was synthesized using standard Fmoc protocols. The mixture was gently shaken at 20 °C for 1 hour. The mixture was filtered using a 50 mL polypropylene filtration tube. The resin was washed with dichloromethane (4 x 10 mL). MEBA (5.25 mmol, 1.24 g) in dichloromethane (2 mL) was added to the resin. The mixture was gently shaken for 20 hours at 20 0C. The resin was washed with dichloromethane (4 x 5 mL). Using method 1, the carbonyl carbon and alpha carbon of the protected residue (glycine) are derived from bromoacetic anhydride, and the backbone nitrogen of the protected residue is derived from MEBA. The synthesis of the peptide chain was then continued using standard Fmoc protocols. Multiple MEB groups can be inserted into a peptide chain using the method described above at multiple sites along the chain.
Method 2. In this method the pre-selected sites of the peptide sequence were modified with a dipeptide unit (see below) in which the amide bond has been protected with a MEB group. The protected dipeptide unit was then coupled to the N-terminus of a growing peptide and the synthesis was continued using standard Fmoc protocols. Preparation of a MEB-protected dipeptide. To a bromoacetic acid pre-loaded HMP-resin (1 mmol, 2.5 g), MEBA (5.2 mmol, 1.23 g) in dichloromethane (10 niL) was added and the mixture was shaken at 20 ° C for 20 hours. The mixture was filtered using a 50 niL polypropylene filtration tube. The resin was washed with dichloromethane (4 x 10 mL). A mixture of N-alpha-Fmoc-protected amino acid (5 mmol) in N,N- dimethylformamide (20 mL), bromotripyrrolidinophosphonium hexafluorophosphate (4.8 mmol, 2.24 g) and N,N-diisopropylethylarnine (10 mmol, 1.74 mL) were mixed for 5 minutes at 20 °C before adding to the resin. The resin was shaken at 20 0C for 18 hours. The resin was then washed with N,N-dimethylformamide (4 x 10 mL) and dichloromethane (2 x 10 mL). The resin was dried in vacuo. The dipeptide was cleaved from the resin with trifluoroacetic acid/water, 9/1 (10.0 mL) at 20 0C for 2 hours. The trifluoroacetic acid-resin mixture was filtered to remove the resin. Trifluoroacetic acid was removed under reduced pressure to give the MEB-protected dipeptide unit. The dipeptide can then be used in SPPS of a longer peptide.
Removal of MEB group from a synthetic peptide
A 50-mL Teflon tube containing a mixture of MEBA-modified peptide (0.018 mmol, 25 mg) and p-cresol (400 mg) was mounted onto an HF apparatus. The tube was immersed into a dry ice acetone bath and anhydrous hydrogen fluoride (10 mL) was condensed. Next, the dry ice acetone bath was replaced by a water bath containing crushed ice, and the reaction was magnetically stirred for 1 hour. The hydrogen fluoride was evaporated from the Teflon tube and trapped into a 15% solution of potassium hydroxide using nitrogen gas at 20 psi for 30 minutes. The Teflon tube was removed from the HF apparatus and the peptide was precipitated with chilled ethyl ether. The solid peptide was then taken up with a 50% solution of acetonitrile-water. The solution was frozen and lyophilized to give the MEB-deprotected peptide.
Removal of backbone nitrogen modifying groups
Table 1 summarizes the results of experiments testing the removal of backbone nitrogen modifying groups under various conditions. Table 1
Synthesis of polypeptide
To test the ability of MEB to prevent peptide aggregation during solid phase peptide synthesis, a peptide fragment with high content of beta sheet secondary structure was prepared. The sequence: CEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLLF AESGQ VYFGIIAL corresponds to the C-terminus of human TNF-alpha. The 48-amino acid peptide was prepared by solid phase peptide synthesis using Fmoc-protected amino acids on an Applied Biosystems 433 A peptide synthesizer according to manufacture specific protocols. A commercial available Fmoc-Leu-Wang Resin (0.47 g. 0.42 mmol/g) was loaded into the reaction vessel and the sequence was extended using five-fold excess of activated Fmoc-amino acids during every coupling step. Fmoc-amino acids were activated by the addition of equimolar amounts of HBTU and HOBt and 2 equivalents of DIEA in DMF. Three MEB groups were introduced at Gl 3, G40 and G45 using method 1. The peptide was cleaved from the resin and deprotected with TFA/EDT/TA/phenol/water/TIPS (68.5:10:10:5:3.5:1 V: V). The TFA resin mixture was filtered. Chilled diethyl ether was added to the filtrate to precipitate the peptide, which was then centrifuged at 2500 RPM for 5 minutes. The pellet was washed three more times with chilled diethyl ether. The peptide was subsequently dissolved in 95% acetic acid and lyophilized. The peptide was purified by reverse-phase HPLC (>98% purity) on a Varian 210 HPLC system with 214-nm UV detection, using a Higgins Analytical C 8 column (2 x 25 cm), a linear gradient of 25- 65% acetonitrile over 45 min, and a flow rate of 5 mL/min in 0.1 % CF3CO2H. The LC-ESI-MS purified peptide showed the correct mass (M+l 6,239.24).
Attempts to make the same 48 amino acid peptide using traditional solid phase peptide synthesis without introducing the three MEB groups at Gl 3, G40 and G45 were unsuccessful.
Other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of making a peptide comprising forming a peptide including a backbone nitrogen modifying group, wherein the backbone nitrogen modifying group includes a substituted aryl group, the substituted aryl group including a directing moiety and a hydrophilic moiety.
2. The method of claim 1 , wherein the peptide is linked to a solid support.
3. The method of claim 1 , wherein the peptide includes at least one commonly occurring natural amino acid residue, wherein the commonly occurring natural amino acid optionally includes a protecting group.
4. The method of claim 1 , wherein the peptide includes at least one non- naturally occurring amino acid residue.
5. The method of claim 1, further comprising adding an amino acid residue to the peptide, thereby extending the peptide.
6. The method of claim 2, further comprising cleaving the peptide from the solid support, wherein cleaving the peptide does not substantially remove the backbone nitrogen modifying group from the peptide.
7. The method of claim 1 , further comprising removing the backbone nitrogen modifying group from the peptide.
8. The method of claim 1, wherein the substituted aryl group is a substituted phenyl group.
9. The method of claim 8, wherein the substituted phenyl group is ortho- unsubstituted.
10. The method of claim 1, wherein the hydrophilic moiety includes a tertiary amine.
11. The method of claim 1 , wherein the peptide is substantially water-soluble.
12. The method of claim 1 , wherein the peptide has the formula:
R7-0 -
wherein:
X is O, S, NH, or a bond; each L1, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O)-, -NRdC(0)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(O)n,-, -NRdSO2-, -SO2NRd-, or -NRd-; each R°, independently, is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re, -O(CRdRe)2NRaRb, -C(O)Ra, -C(O)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(0)0Ra, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl ; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=0)Rb, or halo, wherein R2 is optionally substituted with -iΛR0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each R9, independently, is hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, or halo; each R10, independently, is hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, or halo; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRs, -C(=O)NRfRg, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R5, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group;
R7 is hydrogen, alkyl, aryl, aralkyl, or a solid support;
R8 is hydrogen, alkyl, an amino protecting group, or has the formula -C(=O)CH(R5)R6, wherein R6 is a leaving group; each i, independently, is zero or a positive integer; each j, independently, is zero or a positive integer; k is a positive integer; m is 1 or 2; n is O, 1, 2, 3 or 4; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; z is 1, 2, 3, 4, 5 or 6.
13. The method of claim 12, wherein the peptide includes at least one -L 1 - TRJ C .
14. The method of claim 12, wherein the peptide has the formula:
R7-O- - R8
wherein: X is O, S, NH, or a bond; each L1, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O>, -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(O)1n-, -NRdSO2-, -SO2NRd-, or -NRd-; each Rc, independently, is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb,
-S(O)mORa, -NRdC(O)Re, -0(CRdRe)zNRaRb, -C(O)Ra, -C(0)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo, wherein R2 is optionally substituted with -L'-R0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl., cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRε, -C(=O)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R5, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfR8; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rs, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; R7 is hydrogen, alkyl, aryl, aralkyl, or a solid support; R8 is hydrogen, alkyl, an amino protecting group, or has the formula -C(=O)CH(R5)R6, wherein R6 is a leaving group; each i, independently, is zero or a positive integer; each j, independently, is zero or a positive integer; k is a positive integer; m is 1 or 2; n is O, 1, 2, 3 or 4; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; z is 1, 2, 3, 4, 5 or 6.
15. The method of claim 14, wherein X is O.
16. The method of claim 15, wherein L1 is C1-C4 alkylene.
17. The method of claim 16, wherein Rc is heterocycloalkyl.
18. The method of claim 14, wherein -X-L1 -Rc is 2-(morpholin-4-yl)ethoxy.
19. The method of claim 12, wherein each R5, independently, is hydrogen or alkyl.
20. The method of claim 12, wherein R7 is a solid support and R8 is an amino protecting group.
21. The method of claim 12, wherein each A is C1 alkylene and each R3a is hydrogen.
22. The method of claim 12, wherein the total of all i and all j is less than 300.
23. The method of claim 12, wherein the peptide has a molecular weight of no greater than 40 kDa.
24. The method of claim 12, wherein R8 has the formula -C(=O)CH(R5)R6, wherein R6 is a leaving group.
25. The method of claim 24, wherein each j is zero.
26. The method of claim 24, wherein R6 is halo.
27. The method of claim 24, further comprising contacting the peptide with a compound having the formula:
wherein: X is O, S, NH, or a bond;
L1 is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O)-, -NRdC(O)NRd-3 -OC(O)NRd-, -NRd-C(0)-O, -S, -S(O)1n-, -NRdSO2-, -SO2NRd-, or -NRd-; Rc is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re,
-O(CRdRe)zNRaRb, -C(O)Ra 5 -C(O)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo wherein R2 is optionally substituted with -lΛRc; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl;
R4a is hydrogen, alkyl, or an amino protecting group; m is 1 or 2; n is 0, 1, 2, 3, or 4; and z is 1, 2, 3, 4, 5, or 6.
28. The method of claim 27, wherein the compound is 4-(2-(morpholin-4- yl)ethoxy)benzylamine.
29. The method of claim 28, further comprising adding an amino acid residue to the peptide, thereby forming a longer peptide.
30. The method of claim 12, further comprising contacting the peptide with a compound having the formula:
wherein:
X is O, S, NH, or a bond;
L1 is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(0)NRd-, -NRd-C(O)-, -NRdC(0)NRd-, -OC(O)NRd-, -NRd-C(0)-0-, -S-, -S(O)1n-, -NRdSO2-, -SO2NRd-, or -NRd-;
Rc is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re, -O(CRdRe)zNRaRb, -C(O)R3, -C(O)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(0)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=0)Rb, or halo wherein R2 is optionally substituted with -iΛR0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfR8, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(==NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S;
R4a is hydrogen, alkyl, or an amino protecting group;
R5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, or a leaving group; m is 1 or 2; n is O, 1, 2, 3, or 4; w is O, l, or 2; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; and z is 1, 2, 3, 4, 5, or 6.
31. The method of claim 12, further comprising contacting the peptide with a compound having the formula:
wherein:
X is O, S, NH, or a bond;
L1 is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene,'or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O>, -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(O)1n-. -NRdSO2-, -SO2NRd-, or -NRd-;
Rc is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re, -O(CRdRe)zNRaRb, -C(O)Ra, -C(0)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(0)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo wherein R2 is optionally substituted with -L1 -R0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRs, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S;
R5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfR8, or -NHC(=NH)NRfRg; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, or a leaving group; m is 1 or 2; n is O, 1, 2, 3, or 4; w is 0, 1, or 2; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; and z is 1, 2, 3, 4, 5, or 6.
32. A composition comprising a peptide including a backbone nitrogen modifying group, wherein the backbone nitrogen modifying group includes a substituted aryl group, the substituted aryl group including a directing moiety and a hydrophilic moiety.
33. The composition of claim 32, wherein the peptide includes a plurality of backbone nitrogen modifying groups.
34. The composition of claim 32, wherein the peptide is linked to a solid support.
35. The composition of claim 32, wherein the peptide includes at least one commonly occurring natural amino acid residue, wherein the commonly occurring natural amino acid optionally includes a protecting group.
36. The composition of claim 32, wherein the peptide includes at least one non-naturally occurring amino acid residue.
37. The composition of claim 32, wherein the substituted aryl group is a substituted phenyl group.
38. The composition of claim 32, wherein the substituted phenyl group is ørt/zø-unsubstituted.
39. The composition of claim 32, wherein the hydrophilic moiety includes a tertiary amine.
40. The composition of claim 32, wherein the peptide is substantially water- soluble.
41. The composition of claim 32, wherein the peptide has the formula:
R7-0 - 8
wherein:
X is O, S, NH, or a bond; each L1, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O)-, -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(O)m-, -NRdSO2-, SO2NRd-, or -NRd-; each Rc, independently, is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb,
-S(O)mORa, -NRdC(O)Re, -O(CRdRe)zNRaRb, -C(O)Ra, -C(O)NRdRe, -NRaC(0)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(0)NRaRb, heterocycloalkyl, or
(heterocycloalkyl)alkyl; each Rz, independently, is hydrogen, -Ra, -ORa, -SRa, -NR >aarR>bD, halo wherein R2 is optionally substituted with -IΛRC; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each R9, independently, is hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, or halo; each R10, independently, is hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, or halo; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -C02Rf, halo, haloalkyl, -CN, -NO2, -NRfRg,
-C(=0)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R5, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group;
R7 is hydrogen, alkyl, aryl, aralkyl, or a solid support; R8 is hydrogen, alkyl, an amino protecting group, or has the formula -C(=O)CH(R5)R6, wherein R6 is a leaving group; each i, independently, is zero or a positive integer; each j, independently, is zero or a positive integer, provided that at least one j is a positive integer; k is a positive integer; m is 1 or 2; n is O, I5 2, 3 or 4; and each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; and z is 1, 2, 3, 4, 5 or 6.
42. The composition of claim 41, wherein the peptide includes at least one -
IΛRC.
43. The composition of claim 41, wherein the peptide has the formula:
wherein:
X is O, S5 NH, or a bond; each L1, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O)-, -NRdC(O)NRd-, -OC(O)NRd-, -NRd~C(O)-O-5 -S-, -S(O)1n-,
-NRdSO2-, -SO2NRd-5 or -NRd-; each Rc, independently, is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb 5 (O)mORa, -NRαC(O)Re, -O(CR >dαrR>6e)' aτ>b -S zNRaRD, -C(O)Ra, -C(O)NRαRe, -NRaC(O)RD, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(O)NRaRb, heterocycloalkyl, or
(heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo, wherein R2 is optionally substituted with -I^-R0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN5 -NO2, -NRfRg,
-C(=0)NRfRg, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg,
-C(=0)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O5 and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O5 and S; each R5, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group;
R7 is hydrogen, alkyl, aryl, aralkyl, or a solid support;
R8 is hydrogen, alkyl, an amino protecting group, or has the formula -C(=O)CH(R5)R6, wherein R6 is a leaving group; each i, independently, is zero or a positive integer; each j, independently, is zero or a positive integer; k is a positive integer; m is 1 or 2; n is O, 1, 2, 3 or 4; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; z is 1, 2, 3, 4, 5 or 6.
44. The composition of claim 43, wherein X is O.
45. The composition of claim 44, wherein L1 is C1-C4 alkylene.
46. The composition of claim 44, wherein Rc is heterocycloalkyl.
47. The composition of claim 43, wherein -X-L^R0 is 2-(morpholin-4- yl)ethoxy.
48. The composition of claim 41 , wherein each R5, independently, is hydrogen or alkyl.
49. The composition of claim 41, wherein R7 is a solid support and R8 is an amino protecting group.
50. The composition of claim 41 , wherein R7 is hydrogen and R8 is hydrogen.
51. The composition of claim 41 , wherein each A is C i alkylene and each R3a is hydrogen.
52. The composition of claim 41 , wherein the total of all i and all j is less than 300.
53. The composition of claim 41 , wherein the peptide has a molecular weight of no greater than 40 kDa.
54. The composition of claim 41 , wherein R8 has the formula -C(=O)CH(R5)R6, wherein R6 is a leaving group.
55. The composition of claim 54, wherein R6 is halo.
56. A compound having the formula:
wherein: X is O, S, NH, or a bond;
L1 is C1-C1O alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(O)-, -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(O)1n-, -NRdSO2-, -SO2NRd-, or -NRd-; Rc is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re 5
-O(CRdRe)zNRaRb, -C(O)Ra, -C(O)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo wherein R2 is optionally substituted with -iΛR0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl;
R4a is hydrogen, alkyl, or an amino protecting group; m is 1 or 2; n is 0, 19 2, 3, or 4; and z is 1, 2, 3, 4, 5, or 6.
57. A compound having the formula:
wherein:
X is O, S, NH, or a bond; L1 is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, ~NRd-C(O)-, -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(O)-O-, -S-, -S(O)m-, -NRdSO2-, -SO2NRd-, or -NRd-;
R° is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re, -O(CRdRe)zNRaRb, -C(O)Ra, -C(O)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo wherein R2 is optionally substituted with -LΛR0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg,
-C(=O)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S;
R4a is hydrogen, alkyl, or an amino protecting group;
R5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfRg; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, a solid support, or a leaving group; m is 1 or 2; n is O, 1, 2, 3, or 4; w is O, 1, or 2; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; and z is 1, 2, 3, 4, 5, or 6.
58. A compound having the formula:
wherein:
X is O, S, NH, or a bond;
L1 is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein L1 is optionally interrupted by one or more of -C(O)-, -O-, -C(O)NRd-, -NRd-C(0)-3 -NRdC(O)NRd-, -OC(O)NRd-, -NRd-C(O)-O~, -S-, -S(O)1n-, -NRdSO2-, -SO2NRd-, or -NRd-;
Rc is -NRaRb, -ORa, -SRa, -S(O)mRa, -S(O)2NRaRb, -S(O)mORa, -NRdC(O)Re, -0(CRdRe)2NRaRb, -C(O)Ra, -C(O)NRdRe, -NRaC(O)Rb, -OC(O)NRaRb, -NRdC(O)ORa, -NRdC(O)NRaRb, heterocycloalkyl, or (heterocycloalkyl)alkyl; each R2, independently, is hydrogen, -Ra, -ORa, -SRa, -NRaRb, -NRaC(=O)Rb, or halo wherein R2 is optionally substituted with -L '-R0; each Ra, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, aryl-fused cycloalkyl, aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl, cycloalkenyl-substituted cycloalkyl, or biaryl; each Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each Re, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
(cycloalkyl)alkyl, cycloalkenyl, heterocycloalkyl, (heterocycloalkyl)alkyl, or aryl; each A, independently, is C1-C10 alkylene, alkenylene, alkynylene, cycloalkylene, arylene, or aralkylene, wherein A optionally includes 1-3 heteroatoms selected from N, O and S; each R3, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfR8, -C(=O)NRfRg, or -NHC(=NH)NRfRs; each R3a, independently, is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R3a being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=O)NRfRg, or -NHC(=NH)NRfRg; or R3 and R3a together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S; each R4, independently, is hydrogen or alkyl; or R3 and R4 together with the atoms to which they are attached form a 3-14 membered cyclic, bicyclic or tricyclic moiety, optionally including 1-6 heteroatoms selected from N, O, and S;
R5 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aralkyl, (cycloalkyl)alkyl, (heterocycloalkyl)alkyl or aryl; R5 being optionally substituted with -ORf, -SRf, -CO2Rf, halo, haloalkyl, -CN, -NO2, -NRfRg, -C(=0)NRfRg, or -NHC(=NH)NRfR8; each Rf, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group; each Rg, independently, is hydrogen, alkyl, aryl, aralkyl, or a protecting group;
R11 is hydroxy, alkoxy, aryloxy, aralkyloxy, a solid support, or a leaving group; m is 1 or 2; n is O, 1, 2, 3, or 4; w is O, 1, or 2; each x, independently, is 1, 2, 3, 4, or 5; each y, independently, is 1, 2, 3, 4, or 5; and z is 1, 2, 3, 4, 5, or 6.
59. A method of making a peptide having a predetermined amino acid sequence comprising: determining a beta-sheet-forming propensity for at least a portion of the amino acid sequence; and selecting an amino acid residue of the sequence for modification with a backbone nitrogen modifying group based on the determined beta-sheet-forming propensity.
60. The method of claim 59, wherein the backbone nitrogen modifying group includes a substituted aryl group, the substituted aryl group including a directing moiety and a hydrophilic moiety.
EP06748673A 2005-03-25 2006-03-24 Compounds and methods for peptide synthesis Withdrawn EP1869088A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US66494505P 2005-03-25 2005-03-25
PCT/US2006/010872 WO2006104922A2 (en) 2005-03-25 2006-03-24 Compounds and methods for peptide synthesis

Publications (1)

Publication Number Publication Date
EP1869088A2 true EP1869088A2 (en) 2007-12-26

Family

ID=37053948

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06748673A Withdrawn EP1869088A2 (en) 2005-03-25 2006-03-24 Compounds and methods for peptide synthesis

Country Status (5)

Country Link
US (1) US20090036650A1 (en)
EP (1) EP1869088A2 (en)
CN (1) CN101184779A (en)
BR (1) BRPI0608482A2 (en)
WO (1) WO2006104922A2 (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0441047B1 (en) * 1990-01-19 1996-06-05 Minnesota Mining And Manufacturing Company Thermosettable composition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006104922A2 *

Also Published As

Publication number Publication date
US20090036650A1 (en) 2009-02-05
WO2006104922A3 (en) 2007-04-19
BRPI0608482A2 (en) 2010-01-05
WO2006104922A2 (en) 2006-10-05
CN101184779A (en) 2008-05-21

Similar Documents

Publication Publication Date Title
JP2012502045A (en) Method for producing plumlintide
JP2012525348A (en) Insulin secretion-promoting peptide synthesis using solid phase and solution phase combination techniques
WO2001019849A1 (en) A process for the preparation of h-tyr-d-ala-phe(f)-phe-nh¿2?
US5283293A (en) Reagents for automated synthesis of peptide analogs
JP5985534B2 (en) Indolesulfonyl protecting group for protection of guanidyl and amino groups
KR100241948B1 (en) N alpha-2-(4-nitrophenulsulfonyl)ethoxycarbonyl-amino acids
US6787612B1 (en) Resin derivatization method and uses thereof
WO2006104922A2 (en) Compounds and methods for peptide synthesis
WO1993012076A1 (en) Reagents for automated synthesis of peptide analogs
HU208838B (en) Method for producing peptones containing aza aminoacides by means of solid-phase synthesis
JPH10505575A (en) Method for synthesizing peptidyl argininal
KR100336139B1 (en) Novel peptide active materials and methods for their preparation
Tuchscherer et al. Peptidomimetics for Bridging Structure and Function: Pseudo-Prolines (ΨPro) in Peptide Synthesis, Molecular Recognition, and Drug Design
EP2812345B1 (en) Method of synthesizing peptides, proteins and bioconjugates
US20040101864A1 (en) Chemical process
US5367072A (en) Reagents for automated synthesis of peptide analogs
JP2002521364A (en) Amino-aldehyde solid support, linker therefor, method for producing the same and use thereof
CN108864252B (en) Method for preparing NRX-1074
JP2748897B2 (en) Novel arginine derivative and method for producing peptide using the same
JP2022173143A (en) A drug containing a polypeptide having MMP2 inhibitory activity as an active ingredient
JPS62286978A (en) Oobt esters of amino acids and manufacture
KR19990080857A (en) Liquid 1-Deamino-8-D-Arginine Vasopressin Acetate Synthesis
JPH0525196A (en) Synthesis of cyclic peptide

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20071024

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20090209